Paraffin has long been used as an embedding medium in the preparation of tissue specimens for histological studies. In some instances, plastic resins have also been used as embedding medium. Such embedding processes generally include the steps of specimen fixation (e.g., formalin fixation), dehydration, clearing, paraffin infiltration or impregnation, blocking or embedding in a block of paraffin, slicing the block and specimen into thin sections, mounting the sections on slides, removing the paraffin and solvents employed for this purpose (i.e., dewaxing or deparaffinizing), and staining the sections prior to microscopic analysis. The primary purpose of the embedding medium is to permit the specimens to be sectioned and mounted in the natural state. Paraffin-embedding has the advantage that the wax can be dissolved away from specimens prior to staining, allowing sections to be stained as bare tissue and avoiding the extra difficulties associated with the presence of resin-based embedding medium, which is more difficult to remove.
Recent improvements in paraffin-embedding compositions have broadened its applicability while maintaining its compatibility with downstream preparation and analysis of samples. Consequently, dewaxing of fixed, paraffin-embedded tissue sections is still a widely used methodology, particularly in hospital histopathology laboratories for immunodiagnostic purposes.
One method commonly employed for dewaxing involves the dissolution of the embedding paraffin in organic solvents. For example, xylene, which is a flammable, volatile and toxic organic solvent, is currently commonly used in protocols to solubilize paraffin for dewaxing of specimen sections. Typically, the microscope slide-mounted specimen is immersed in a xylene bath until the paraffin is dissolved.
Another method commonly employed for dewaxing involves the melting and removal of embedding paraffin in a heated bath containing an aqueous buffer solution. The use of heated buffer solution has the additional advantage that it allows antigen retrieval through a process known as heat-induced epitope retrieval (“HIER”). HIER is a pretreatment procedure often used prior to immunohistochemistry (“IHC”) or in-situ hybridization (“ISH”) procedures to improve staining by heat-induced modification of the molecular conformation of target proteins contained in slide-mounted specimen material. Typically, this modification process is necessary because, although aldehyde-based fixatives (e.g., formalin) are excellent for preserving cellular morphology, they also cause protein cross-linking, resulting in the inability of some protein epitopes to bind complementary antibodies. HIER is commonly used in conjunction with enzyme digestion as a means of improving the reactivity of various antigens within IHC/ISH staining reactions.
Existing buffer formulations that are configured to simultaneously dewax slides and perform HIER prior to IHC/ISH utilize small concentrations of surfactants and other emulsifiers to break up paraffin. The HIER procedure places slides at an elevated temperature (e.g., from 70 to 100° C.), allowing paraffin to melt and surfactants to gently lift paraffin to the buffer surface. However, known buffer formulations and methods have limited performance, sometimes creating paraffin streaking, slide recoating, and inconsistent staining results.
Accordingly, there is a need for dewaxing compositions and methods that can effectively remove paraffin or improved paraffin-based embedding materials from specimens prior to immunohistochemical or other diagnostic analyses, while minimizing danger to users, allowing compatibility with automated systems, and maintaining compatibility with downstream analyses. Dewaxing compositions and methods that entail no or limited toxicity or carcinogenicity, produce no or minimal odors, reduce the quantity of toxic solvents used, minimize hazardous wastes, and/or decrease corrosiveness and flammability are needed.